1. Field of Invention
The present invention relates to a method for suppressing nonspecific extension of primers in the primer extension method.
2. Background Art
The primer extension reaction, which forms a nucleic acid complementary to a nucleic acid template, not only has an important role in an organism but is also used as an essential technique in genetic engineering. In the reaction, a template and a primer which is complementary to the template form a double strand, and then polymerase adds the mononucleotides complementary to the template at the 3' end of the primer. Vary et al. proposed a method of detecting nucleic acids using the above template-dependent primer extension reaction (U.S. Pat. No. 4,851,331).
Various gene amplification methods utilizing the above primer extension method (for example, the PCR method, SDA method, RCR method, NASB method, 3SR method: Keller, G. H. et al., DNA Probes, pp. 255-297, Stockton Press (1993); Persing, D. H. et al., Diagnostic Molecular Microbiology, pp. 51-87, American Society for Microbiology (1993)) have been developed, which have significantly contributed to the advancement of genetic engineering.
However, in these gene amplification methods using the primer extension method, excessive amounts of reagents (polymerase, primer, unit nucleic acid, etc.) relative to a template gene to be amplified are necessary in the early stages of the reaction in order to amplify the gene by about million times. Therefore, nonspecific hybridizations are very likely to occur (Chou et al., Nucleic Acids Res., 20, 1717 (1992)). Furthermore, even in the PCR method which is believed to be the best in terms of specificity, it is difficult to perform highly specific amplification when the amount of template nucleic acid is extremely small, or the presence of large quantities of impurities derived from a sample. Also, primer dimers were found to cause an unexpected problem in the PCR method (Li et al., Proc. Natl. Acad. Sci. USA. 87, 4580-4584 (1990)).
To solve the above problems, the Hot Start Method (Chou et al., Nucleic Acids Res. 20, 1717-1723 (1992)), a method using a polymerase antibody (Kellogg et al., Bio Techniques 16, 1134-1137 (1994); Sharkey et al., BIO/TECHNOLOGY 12, 506-507 (1994)), and others have been proposed. However, none of these methods can sufficiently suppress nonspecific hybridization nor nonspecific extension reaction after the start of the PCR reaction, and cannot be applied to any other amplification method except the PCR method. A nested PCR method using 2 sets of primers (Pierre et al., J. Clin. Microbiol. 29, 712-717 (1991)) definitely improves specificity; however, this method is considered to be procedurally unpractical.